Collagen Bundles Research Articles

SP34. Development of Sutureless Vaso-Lock for Vascular Anastomosis

PURPOSE: Micro- and macrovascular anastomoses involve suturing together of blood vessels, which is a critical foundational surgical skill. However, it faces many challenges, such as decade-long training and inherent dexterity of the surgeon. Procedures are long and expensive, require specialized operating rooms and equipment, making them prohibitive in many hospitals. Our team has created a unique anastomotic device, Vaso-Lock, as a sutureless coupler. Vaso-Lock is intraluminal, holding free vessel ends together with traction by anchors. We aim to apply Vaso-Lock to change the paradigm of surgical training and practice, and improve technical capabilities for vascular anastomosis. METHODS: We have utilized 3D-printing to prototype Vaso-Locks with quick design adjustments. Vaso-Lock maintained its intact structure after ethylene oxide sterilization, which was evaluated by scanning electron microscope. Vaso-Lock was deployed in a porcine model for anastomosis of common carotid artery and internal jugular vein. Doppler ultrasound was used to longitudinally assess patency, blood flow velocities, and lumen preservation. Histological studies were performed to evaluate endothelialization and device-vessel interface biocompatibility. RESULTS: Vaso-Lock maintained anastomosis of common carotid artery to internal jugular vein in 3 porcine, with no evidence of leakage after deployment. Doppler ultrasonography demonstrated the mixture of arterial and venous blood inside the Vaso-Lock. During the extraction of Vaso-Lock, no blood clots were identified at the anastomosis site. Guidewire went through the explanted blood vessels anastomosed by the Vaso-Lock without resistance. In terms of histological analysis, we performed four types of staining: H&E, Masson’s Trichrome (blue for collagen), Verhoeff-Van Gieson (black for elastin), and Alizarin Red (red for calcium). All tissue sections with Vaso-Locks displayed open lumen at the anastomosis site. Inflammation cells, along with collagen bundles, appeared around anchors of the Vaso-Lock. Calcium was identified around the Vaso-Lock at 2-week. Intimal growth occurred under the Vaso-Lock at 6-week. We are examining the effect of Vaso-Lock on cellular structure of the blood vessel by immunohistochemistry. We are also evaluating heparinized Vaso-Lock in terms of its long-term patency and biocompatibility. CONCLUSIONS: Vaso-Lock maintained patency at least 6 weeks for arteriovenous anastomosis along with a natural healing response in a porcine model. This novel device will dramatically enhance the efficiency with an assortment of micro- and macrovascular anastomoses ranging from tissue flaps to arteriovenous fistula creations, making anastomosis globally available.

Histopathological Study on Collagen in Full-Thickness Wound Healing in Fraser's Dolphins (Lagenodelphis hosei).

Fraser's dolphins (Lagenodelphis hosei) possess great healing abilities. Their skin composition can be restored after wounding, including collagen spacing, orientation, and bundle thickness. However, it remains unclear how collagens are involved in the wound-healing process and eventually regain normality in Fraser's dolphins. Learned from the other two scarless healing animals, changes in type III/I collagen composition are believed to modulate the wound healing process and influence the scarring or scarless fate determination in human fetal skin and spiny mouse skin. In the current study, Herovici's, trichrome, and immunofluorescence staining were used on normal and wounded skin samples in Fraser's dolphins. The results suggested that type I collagens were the main type of collagens in the normal skin of Fraser's dolphins, while type III collagens were barely seen. During the wound healing process, type III collagens showed at early wound healing stages, and type I collagen increased in the mature healed wound. In an early healed wound, collagens were organized in a parallel manner, showing a transient hypertrophic-like scar, and eventually restored to normal collagen configuration and adipocyte distribution in the mature healed wound. The remarkable ability to remove excessive collagens merits further investigation to provide new insights into clinical wound management.

Collagens Regulating Adipose Tissue Formation and Functions.

The globally increasing prevalence of obesity is associated with the development of metabolic diseases such as type 2 diabetes, dyslipidemia, and fatty liver. Excess adipose tissue (AT) often leads to its malfunction and to a systemic metabolic dysfunction because, in addition to storing lipids, AT is an active endocrine system. Adipocytes are embedded in a unique extracellular matrix (ECM), which provides structural support to the cells as well as participating in the regulation of their functions, such as proliferation and differentiation. Adipocytes have a thin pericellular layer of a specialized ECM, referred to as the basement membrane (BM), which is an important functional unit that lies between cells and tissue stroma. Collagens form a major group of proteins in the ECM, and some of them, especially the BM-associated collagens, support AT functions and participate in the regulation of adipocyte differentiation. In pathological conditions such as obesity, AT often proceeds to fibrosis, characterized by the accumulation of large collagen bundles, which disturbs the natural functions of the AT. In this review, we summarize the current knowledge on the vertebrate collagens that are important for AT development and function and include basic information on some other important ECM components, principally fibronectin, of the AT. We also briefly discuss the function of AT collagens in certain metabolic diseases in which they have been shown to play central roles.

Bioinspired Strong and Tough Organic–Inorganic Hybrid Fibers

High‐strength and high‐toughness bio‐based fibers attract broad interest in biomechanical applications. Herein, strong and tough organic–inorganic regenerated silk fibroin/hydroxyapatite (RSF/HAP) hybrid fibers are prepared using a single‐channel microfluidic device. Calcium phosphate oligomers (CPOs) dispersed in the RSF matrix first grow into spherical amorphous calcium phosphates (ACPs), which then crystallize into needle‐like HAPs under a humidity condition, mimicking the biomineralization in collagen bundles. HAPs are better aligned along the RSF/HAP fiber direction after poststretching, forming a highly ordered and densely packed microstructure within the fiber and thus facilitating highly dense noncovalent interactions between rigid inorganic HAP nanocrystals and flexible organic RSF matrix. The highly dense noncovalent interactions endow the organic–inorganic hybrid fibers with superior mechanical properties and twisted RSF/HAP fiber bundles demonstrate a remarkable tensile strength of 778 MPa, a high Young's modulus of 17.8 GPa, a large tensile strain of 19.9%, and an excellent toughness of 121 MJ m−3 after proper twisting treatments. RSF/HAP hybrid fibers also show good performances against static loading, dynamic impact, and extreme cold condition and they can maintain their mechanical properties down to −50 °C. Therefore, the fibers are strong and tough and the strategy is facile and efficient.

Spindle Cell Lipoma and Pleomorphic Lipoma: An Update and Review.

Spindle cell lipoma (SCL) is a benign adipocytic tumor that primarily occurs in the subcutis of the posterior neck, upper back, and shoulder, particularly of middle-aged males. SCL and pleomorphic lipoma (PL) represent a morphological spectrum of one disease process. The lesion typically presents as a relatively small (<5 cm), mobile, slow-growing, painless mass. Magnetic resonance imaging reveals the lesion to be a well-defined subcutaneous mass with a mixture of adipose and non-adipose components. Intense enhancement of the non-adipose component is seen after contrast administration. Histologically, SCL is composed of variable distributions of mature adipocytes, bland spindle cells and ropey collagen bundles and PL also contains pleomorphic and multinucleated floret-like giant cells. By immunohistochemistry, the spindle and pleomorphic/floret-like giant cells of SCL/PL are diffusely positive for CD34 and show loss of nuclear RB transcriptional corepressor 1 (RB1) expression. Recent cytogenetic and molecular genetic studies have shown heterozygous deletions of 13q14, including the RB1 gene. SCL/PL can be successfully treated with simple excision, with a very low recurrence rate. Knowledge of these peculiar tumors is important because it can mimic a variety of benign and malignant soft-tissue tumors. This review provides an updated overview of the clinical, radiological, histopathological, cytogenetic, and molecular genetic features of SCL/PL.

P162 Biosamples from VEDOSS patients show classic pathological signs of Scleroderma:Opportunity for a biological diagnosis of disease

Abstract Background/Aims The VEDOSS study has recently indicated that more than 50% of patients affected by Raynaud’s phenomenon (RP) and specific SSc anti-nuclear antibodies (ANA) and/or nailfold videocapillaroscopy changes (NVC) (sRP) satisfied ACR/EULAR 2013 criteria within 5 years, indicating a window of opportunity for early detection of SSc. Here we aimed to determine whether sera, skin or dermal fibroblasts cultured from VEDOSS patients showed any biological hallmark of SSc. Methods VEDOSS patients were enrolled in the national inception cohort for patients at risk of Scleroderma (Kennedy Cohort, n = 64) . Sera were tested for IFN-inducible chemokines and biomarker of extracellular matrix turnover (ELF test) and compared to Healthy control (HC) and SSc sera (n = 56, 64, respectively). 3mm skin punch biopsies were taken from the forearms of 12 VEDOSS patients, and 5 healthy controls and 6 SSc patients. Biopsies were analysed for haematoxylin and eosin (H&amp;E), masson trichrome staining (MT) and immunohistological (IHC) staining for CD45. A dermatopathologist blinded to clinical features undertook the analysis. A probabilistic image analysis model was used to detect areas of immunopositivity (CD45) and collagen staining (MT) within the samples, using HeteroGenius Medical Imaging Manager colour analysis. In addition, 7 biopsies along with 2 healthy control and 3 SSc, were used to explant fibroblasts cultures. mRNA and protein were isolated from primary fibroblasts and processed for RT-qPCR and western blotting analyses. Results Skin biopsies from VEDOSS patients showed evidence of fibrosis and increased collagen bundles within the dermis evidenced in H&amp;E and MT staining, as usually seen in SSc, compared to HC. IHC showed increased number of CD45+ cells in VEDOSS. Semiquantitative analysis of histopathological samples indicated a significant correlation between MT and CD45 staining for VEDOSS (R = 0.8828 P = 0.0016). In vitro, fibroblasts from VEDOSS patients showed an average 5-fold higher collagen mRNA levels compared to HC fibroblasts, at a similar range to that seen in SSc fibroblasts, confirmed at protein level via western blotting. Sera from VEDOSS patients showed significantly higher IFN-inducible chemokines compared to HC and an intermediate levels compared to SSc [Mean (STDV) HC = 4.69(0.29) vs. VEDOSS= 4.93 (0.39); SSc= 5.30 (0.54)]. ELF score was within normal range for most patients with SRP and showed no statistical difference to HC. Conclusion Although pilot in nature, this study suggests that patients with no clinical signs of skin fibrosis in their forearms already show biomarker signs of SSc both in their sera, skin biopsies and fibroblast level. Our data indicate that skin thickening is a late manifestation of SSc pathogenesis and early window of opportunity of patients with VEDOSS could be targeted for immune intervention and antifibrotic intervention. Disclosure R.L. ross: None. E. Clarke: None. W. Merchant: None. I. Georgiou: None. P. Mulipa: None. A. Carriero: None. G. Abignano: None. A. herrick: None. C. Denton: None. N. Riobo-Del Galdo: None. F. Del Galdo: Consultancies; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.

Isolation and Biochemical Properties of Type II Collagen from Blue Shark (Prionace glauca) Cartilage

Numerous studies have shown that type II collagen (CII) has a potential role in the treatment of rheumatoid arthritis. However, most of the current studies have used terrestrial animal cartilage as a source of CII extraction, with fewer studies involving marine organisms. Based on this background, collagen (BSCII) was isolated from blue shark (Prionace glauca) cartilage by pepsin hydrolysis and its biochemical properties including protein pattern, total sugar content, microstructure, amino acid composition, spectral characteristics and thermal stability were further investigated in the present study. The SDS-PAGE results confirmed the typical characteristic of CII, comprising three identical α1 chains and its dimeric β chain. BSCII had the fibrous microstructure typical of collagen and an amino acid composition represented by high glycine content. BSCII had the typical UV and FTIR spectral characteristics of collagen. Further analysis revealed that BSCII had a high purity, while its secondary structure comprised 26.98% of β-sheet, 35.60% of β-turn, 37.41% of the random coil and no α-helix. CD spectra showed the triple helical structure of BSCII. The total sugar content, denaturation temperature and melting temperature of BSCII were (4.20 ± 0.03)%, 42 °C and 49 °C, respectively. SEM and AFM images confirmed a fibrillar and porous structure of collagen and denser fibrous bundles formed at higher concentrations. Overall, CII was successfully extracted from blue shark cartilage in the present study, and its molecular structure was intact. Therefore, blue shark cartilage could serve as a potential source for CII extraction with applications in biomedicine.

The Therapeutic Effects of Autologous Platelet-Rich Plasma Gel on Cutaneous Wound Healing in Rescued Horses

This study explored the effect of platelet-rich plasma gel on skin wound healing in rescued horses. A total of twenty horses were used and allocated into two groups: PRP-treated Group A (n=10) and ASD-treated Group B (n=10). Group A animals were treated with autologous PRP gel, while group B animals were treated with sterile saline. A full-thickness (1.5 mm2) skin wounds were selected in each horse. Histopathological examination of wound tissue in horses was performed on days 8, 40, and 60. Wound epithelialization was assessed by HE staining, and collagen re-establishment was assessed by Masson’s trichrome staining. The CAT activity and MDA concentration were assessed in blood samples on days 8, 40, and 60. All data were subjected to statistical analysis. We observed a highly significant increase (P&lt;0.01) in re-epithelization at days 40 to 60 in the PRP wounds compared to the ASD wounds. Collagen was well organized (P&lt;0.01) in the PRP wounds compared with the ASD wounds at day 40 to day 60. MDA concentration was significantly decreased from day 40 to day 60 (P&lt;0.05) in the PRP wounds than the ASD wounds. CAT activity showed no difference (P&gt;0.05) between PRP wounds and ASD wounds. In conclusion, compared to ASD wounds, PRP wounds promoted cutaneous wound healing in horses by suppressing oxidative stress levels, accelerating wound epithelialization, and generating organized tissue, interlocking collagen bundles of dermal collagen. However, ultrasonographical assessment level study is needed to further investigate the effect of PRP gel on skin wound healing in rescued horses.&#x0D; Keywords: Horses, Oxidative stress, Platelet-Rich Plasma, Sterile saline, Skin Wound Healing.

Ex vivo demonstration of canine corneal pre-Descemet’s anatomy using pneumodissection as for the big bubble technique for deep anterior lamellar keratoplasty

The recent discovery and characterization of pre-Descemet’s layer (PDL; also termed the Dua’s layer or the Dua-Fine layer) has advanced the understanding of various posterior corneal pathologies and surgeries in human. This study aimed to characterize the ultrastructure of the posterior stroma and interfacial zone of Descemet’s membrane (DM) in canine eyes. Eighteen canine corneo-scleral discs were included. Intrastromal air injection resulted in the formation of type 1 big bubble (BB) in 73% (n = 11/15) of corneas, with a mean diameter of 11.0 ± 1.3 mm. No type 2 BB was created. Anterior segment optical coherence tomography, histology and transmission electron microscopy confirmed that the wall of BB was composed of DM, in contact with remaining stroma (canine PDL; cPDL). The cPDL was populated with keratocytes, of varying thickness of 16.2 ± 4.2 µm in close apposition to the DM, and composed of collagen bundles arranged in transverse, longitudinal and oblique directions. The interfacial zone, between DM and cPDL, showed fibril extension in all three directions, predominantly longitudinal. Irregular extensions of DM material into cPDL stroma were observed. No long-spaced collagen was detected. In conclusion, there exists a well-defined cleavage plane between the posterior stroma and cPDL, with similar but not identical characteristics as in humans, that is revealed by pneumodissection. This adds to our understanding of the anatomy of the posterior most canine cornea, which will have significant clinical impact on posterior corneal surgery and understanding of corneal pathology in dogs.

Lamprey eyes change across time

Lampreys are jawless fishes known for their parasitic lifestyles. Young lampreys move from freshwater streams to find a host to latch onto in the ocean. After more than a year of feeding on a host, the lampreys move back to freshwater streams to breed and eventually die. These very different phases of their lifestyle come with major differences in their environments, moving from saltwater to freshwater, and going from eating continuously to living off energy reserves in the body. It is not surprising that these big differences in the environment might result in changes to the lamprey's body, including how they sense the world around them. Previous work has suggested that the sizes of different parts of the brain responsible for the senses change with age. So how might vision change when a lamprey moves from a well-fed state in a marine environment to starving in a freshwater stream? Hermann Collin from the University of New South Wales, Australia, and colleagues from La Trobe University, Australia, and the University of Western Australia studied lamprey eye anatomy in the early and late stages of the life cycle to understand how vision might change across time.The researchers collected Australian lampreys (Mordacia mordax) from several streams in New South Wales, Australia, at two points in the animals’ life cycle – the early phase when they were younger and smaller, moving downstream towards the sea; and the late phase, when they were older, larger and moving upstream away from the sea and back to their freshwater breeding grounds. The researchers used powerful microscopes to compare the anatomy of the eyes, including the cornea (the clear part of the eye that allows light in) at the two different life stages.They discovered that there were several differences between the eyes of lampreys in the early life phase and those in the late phase. The outer layers of the cornea were thicker in the late life stage lampreys than the younger lampreys that were headed out to sea. These thicker layers of the cornea in older lampreys have a similar refractive index to sea water, so they do not bend light as it enters the eye, giving the fish a clear view; the cornea is optimized for vision in the saltwater that the older lampreys migrated from. The team also investigated the mucoid layer of the cornea, which is responsible for keeping the eye moist. They discovered that it is attached to the tissue that surrounds the iris in the late phase of the lamprey's life, but is detached in the young lampreys, which may allow extra room for the eye to grow when fish are young, similar to how human babies are born with unfused skulls to allow for brain growth. The young lampreys also had less organized collagen bundles in the center of the cornea compared with the older lampreys. As the lamprey grows, these collagen bundles likely become more organized to keep the cornea as transparent as possible, which helps the lamprey to see which fishes may serve as their next buffet.While lampreys aren't particularly charismatic, they offer some amazing insights into the eyes and vision of some of the earliest vertebrate ancestors, as they are so closely related to these ancient animals. Understanding how their vision works and how it changes when exposed to different environments can help us to uncover how modern vertebrate vision originated and how vision may be affected by aging or the environment. This knowledge can help researchers to better understand how perception may have evolved and how senses such as vision can respond to changes in challenging environments.

Deep Learning for Microstructural Characterization of Synchrotron Radiation-Based Collagen Bundle Imaging in Peri-Implant Soft Tissues

The study of the organizational kinetics in the area surrounding the transmucosal part of dental implants promises to ensure an accurate diagnosis of the healing process, in terms of osseointegration and long-term implant success. In this demonstrative work, the morphological, qualitative and quantitative characteristics of 3D images of collagen bundles obtained by synchrotron-based high-resolution X-ray tomography were analyzed. Data analysis was performed using deep learning algorithms, neural networks that were applied on multiple volumes extracted from connective portions of different patients. The neural network was trained with mutually consistent examples from different patients; in particular, we used a neural network model, U-Net, well established when applying deep learning to datasets of images. It was trained not only to distinguish the collagen fibers from the background, but also to subdivide the collagen bundles based on the orientation of the fibers. In fact, differently from conventional thresholding methods, deep learning semantic segmentation assigns a label to each pixel, not only relying on grey level distribution but also on the image morphometric (shape or direction) characteristics. With the exception of Pt2 biopsies that, as confirmed by the polarized light investigation, were shown to present an immature tissue condition, the quantity, the anisotropy degree and the connectivity density of transverse bundles were always demonstrated to be higher than for longitudinal ones. These are interesting and new data; indeed, as collagen bundles are organized in an intertwining pattern, these morphometric and 3D complexity parameters, distinguished in transversal and longitudinal directions, give precise indications on the amount and distribution of connective tissue forces exerted during the healing process.

CD248 Regulates Inflammation and Encapsulation in Silicone-Related Capsule Formation.

Capsular contracture is the most common reason for having a secondary breast implant operation. The failure of the implanted device and discomfort are related to foreign body response, which involves a pathologic encapsulation. An up-regulated expression of CD248 was previously demonstrated to modulate inflammation and fibrosis. The authors hypothesized that CD248 contributes to foreign body reaction and contracture during silicone-stimulated capsule formation. A murine capsular contracture model was established to correlate CD248 with capsular contracture. The timing and site of CD248 expression were characterized by protein analysis and histologic examination. The capsules between wild-type mice and CD248 knockout mice were compared in this model to verify the possible role of CD248 in silicone-related capsule formation. CD248 was expressed in the peri-silicone implant capsule by stromal fibroblast and perivascular fibroblast. CD248 was overexpressed on day 4 and down to a constant level, but it was still up-regulated through day 21 to day 56 after silicone implantation. The CD248 knockout mice showed a prolonged inflammation period, whereas the wild-type mice developed a thinner but more collagenous capsule. In conclusion, an effective murine capsular contracture model was established to study the relationship between CD248 and capsular contracture. CD248 may play a role in inflammation and encapsulation during silicone implantation. CD248 deletion in mice contributed to a loose and irregular collagen bundle in a capsule area, implying a decrease in contracture. Therefore, CD248 could be a potential therapeutic target in capsular contracture. CD248 may play a role in inflammation and encapsulation during silicone implantation. It could be a potential therapeutic target in clinical capsular contracture.

The first record of fossilized soft parts in ossified tendons and implications for the understanding of tendon mineralization

AbstractPreservation of soft parts (collagen fibres, blood vessels and cells) in extinct vertebrates is rare and usually limited to fossilized bone and cartilage. Well-preserved coarse collagenous fibre bundles embedded in a mineralized matrix of tendons, as well as numerous hollow, tubular structures consistent morphologically with fibril bundles, blood vessels and associated cells, were identified in ossified tendons of Late Cretaceous ornithischians from North America and Central East Asia. Detailed, high-accuracy imaging, along with spectroscopic characterization of those fibrous structures and comparison with ossified tendons of modern-day turkeys, support the proposition that physiologically driven tendon ossification is common for avians and non-avian dinosaurs. The examined soft parts were preserved through the pathway of iron-induced crosslinking and alumino-silification, documenting a variety of pathways for the preservation of soft parts, depending on the burial environment. For the first time, the structure of dinosaur fossilized tendons is analysed in detail, revealing shared histogenetic principles with modern birds and the nature of preservation.

Degenerated lumbricals in the feet of adult human cadavers: case series.

In the foot, the lumbricals flex the metatarsophalangeal joints and extend the interphalangeal joints. The lumbricals are known to be affected in neuropathies. It is not known whether they may degenerate in normal individuals. Here, we report our findings of isolated degenerated lumbricals in seemingly normal feet of two cadavers. We explored lumbricals in 20 male and 8 female cadavers that were 60-80 years of age at the time of death. As part of routine dissection, we exposed the tendons of the flexor digitorum longus and the lumbricals. From the degenerated lumbricals, we took some tissue for paraffin-embedding, sectioning, and staining by hematoxylin and eosin, and Masson's trichrome technique. Of the 224 lumbricals studied, we found four apparently degenerated lumbricals in two male cadavers. In the first, the 2nd and 4th lumbricals in the left foot and the 2nd in the right foot were degenerated. In the second, the right 4th lumbrical was degenerated. Microscopically, the degenerated tissue was made of bundles of collagen. The lumbricals may have degenerated due to compression of their nerve supply. We cannot comment on whether the functionality of the feet were affected by these isolated degeneration of the lumbricals.

Histological and histomorphometric study of human palatal mucosa: Implications for connective tissue graft harvesting.

To analyse the histological structure and histomorphometric characteristics of human hard palatal mucosa in order to determine the donor site of choice for connective tissue grafts from a histological point of view. Palatal mucosa samples from six cadaver heads were harvested at four sites: incisal, premolar, molar and tuberosity. Histological and immunohistochemical techniques were performed, as was histomorphometric analysis. In the current study, we found that the density and size of cells were higher in the superficial papillary layer, whereas the thickness of the collagen bundles increased in the reticular layer. Excluding the epithelium, the mean percentage of lamina propria (LP) and submucosa (SM) was 37% and 63%, respectively (p < .001). LP thickness showed similar values in the incisal, premolar and molar regions, and a significantly greater thickness in tuberosity (p < .001). The thickness of SM increased from incisal to premolar and molar, disappearing in the tuberosity (p < .001). As dense connective tissue of LP is the tissue of choice for connective tissue grafts, the best donor site from a histological point of view is tuberosity because it is composed only of a thick LP without the presence of a loose submucosal layer.

Cross-Polarization Optical Coherence Tomography for Clinical Evaluation of Dermal Lesion Degrees in Vulvar Lichen Sclerosus.

The study included 10 patients without pathology and 39 patients with VLS diagnosed histologically. CP OCT was performed in vivo on the inner surface of the labia minora, in the main lesion area. From each scanning point, a 3.4×3.4×1.25-mm3 3D data array was obtained in 26 s. CP OCT examination results were compared with histological examination of specimens stained with Van Gieson's picrofuchsin.Quantitative analysis of OCT images was performed by measuring the attenuation coefficient in co-polarization and cross-polarization. For visual analysis, color-coded charts were developed based on OCT attenuation coefficients. According to histological examination, all patients with VLS were divided into 4 groups as per dermal lesion degree: initial (8 patients); mild (7 patients); moderate (9 patients); severe (15 patients). Typical features of different degrees were interfibrillary edema up to 250 μm deep for initial degree, thickened collagen bundles without edema up to 350 μm deep for mild degree, dermis homogenization up to 700 μm deep for moderate degree, dermis homogenization and total edema up to 1200 μm deep for severe degree.Pathological processes in dermis during VLS like interfibrillary edema and collagen bundles homogenization were visualized using CP OCT method based on values of attenuation coefficient in co- and cross-polarization channels. However, CP OCT method appeared to be less sensitive to changes of collagen bundles thickness not allowing to distinguish thickened collagen bundles from normal ones with enough statistical significance. The CP OCT method was able to differentiate all degrees of dermal lesions among themselves. OCT attenuation coefficients differed from normal condition with statistical significance for all degrees of lesions, except for mild. For the first time, quantitative parameters for each degrees of dermis lesion in VLS, including initial degree, were determined by CP OCT method allowing to detect the disease at an early stage and to monitor the applied clinical treatment effectiveness.

High-Performance Polarization Microscopy Reveals Structural Remodeling in Rat Calcaneal Tendons Cultivated In Vitro.

Collagenous tissues exhibit anisotropic optical properties such as birefringence and linear dichroism (LD) as a result of their structurally oriented supraorganization from the nanometer level to the collagen bundle scale. Changes in macromolecular order and in aggregational states can be evaluated in tendon collagen bundles using polarization microscopy. Because there are no reports on the status of the macromolecular organization in tendon explants, the objective of this work was to evaluate the birefringence and LD characteristics of collagen bundles in rat calcaneal tendons cultivated in vitro on substrates that differ in their mechanical stiffness (plastic vs. glass) while accompanying the expected occurrence of cell migration from these structures. Tendon explants from adult male Wistar rats were cultivated for 8 and 12 days on borosilicate glass coverslips (n = 3) and on nonpyrogenic polystyrene plastic dishes (n = 4) and were compared with tendons not cultivated in vitro (n = 3). Birefringence was investigated in unstained tendon sections using high-performance polarization microscopy and image analysis. LD was studied under polarized light in tendon sections stained with the dichroic dyes Ponceau SS and toluidine blue at pH 4.0 to evaluate the orientation of proteins and acid glycosaminoglycans (GAG) macromolecules, respectively. Structural remodeling characterized by the reduction in the macromolecular orientation, aggregation and alignment of collagen bundles, based on decreased average gray values concerned with birefringence intensity, LD and morphological changes, was detected especially in the tendon explants cultivated on the plastic substrate. These changes may have facilitated cell migration from the lateral regions of the explants to the substrates, an event that was observed earlier and more intensely upon tissue cultivation on the plastic substrate. The axial alignment of the migrating cells relative to the explant, which occurred with increased cultivation times, may be due to the mechanosensitive nature of the tenocytes. Collagen fibers possibly played a role as a signal source to cells, a hypothesis that requires further investigation, including studies on the dynamics of cell membrane receptors and cytoskeletal organization, and collagen shearing electrical properties.

Mammary myofibroblastoma: a case report

Background: Myofibroblastoma is a benign mesenchymal tumour which can be challenging to diagnose. It has a range of histological features that overlap with other benign or malignant neoplasms.1 Adjunct testing that demonstrates RB1 loss on immunohistochemistry and monoallelic loss of 13q14 region on FISH can seal the diagnosis of this lesion which is curative with local excision.2,3 Case summary: We report a case of a 45-year-old female who presented with a right breast nodule. A core biopsy showed a stromal spindle cell proliferation and the working diagnosis included myoid hamartoma, fibroepithelial lesion and a spindle cell carcinoma. The excision of the lesion revealed a vaguely circumscribed spindle cell proliferation with spindle cells arranged in fascicles interspersed by hyalinised collagen bundles. The tumour cells were positive on immunohistochemistry for SMA, Desmin and ER but negative for CKAE1/3, B-catenin, S100, CK broad spectrum and p63. It also showed a loss of RB1. The FISH analysis showed a loss of RB1 at 13q14 in 35.5% of the nuclei confirming the diagnosis of a benign myofibroblastoma. Conclusion: The constellation of histological features when supported with monoallelic loss of 13q14 region and RB1 loss on immunohistochemistry confirms the diagnosis of this benign entity preventing unnecessary further management.

Efficacy and safety of microbial transglutaminase-induced scleral stiffening invivo

The purpose of this study was to investigate the efficacy and safety of microbial transglutaminases (mTGases) during scleral collagen cross-linking (CXL) in vivo. Sixteen New Zealand white albino rabbits were treated with sub-Tenon's injections of 2 ml of 1 U/ml mTGases in the right eye and 2 ml of phosphate buffer saline (PBS) in the left eye. The rabbits were killed 2 weeks after the injection, and all eyeballs, including some scleral strips, were processed. The elastic modulus was measured with a biomaterials tester. Histopathological analysis and transmission electron microscopy (TEM) were used for the morphological observations. The elastic modulus of the mTGase-treated sclera was 15.79 ± 2.93 MPa, and that of the control was 6.91 ± 2.23 MPa, indicating an increase of 129% after the mTGases treatment (P < 0.05). The density of the scleral collagen bundles and diameter of the collagen fibrils increased compared with those in the control group. No apoptosis was detected in the retina or posterior sclera by TUNEL staining, and no histological damage was observed on the TEM scan. This study is based on a short-term study on animal models. These results indicate that mTGase-mediated scleral CXL is a promising approach to effectively stiffen the sclera and safe enough for retina, and may be a useful treatment modality for strengthening scleral tissue.

Establishment of a Beagle Dog Model of Dry Eye Disease.

To establish a Beagle dog model of dry eye disease (DED). DED models were induced by surgical removal of orbital lacrimal glands and entire resection of third eyelids in the left eyes of six Beagle dogs. Intact right eyes served as self-controls. Non-anaesthetized Schirmer test (STT), tear break-up time (TBUT), and fluorescein staining grading were performed monthly after operation. Interleukin (IL)-1β, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) levels were detected in tears and conjunctiva tissues. Six months after surgery, conjunctiva and cornea were collected and histopathologically analyzed. Signs of DED appeared within one month after surgery and then remained stable. STT values were significantly reduced by 88% within 3 weeks after operation and remained stable over months with 1.6 ± 0.4 mm. Mean TBUT decreased significantly within two months after operation and maintained 5.2 ± 1.1 seconds. The mean fluorescein staining score was highest at the first month and then was reduced, eventually reaching a balance with 11.0 ± 1.3 points. Elevated levels of IL-1β, IL-8, IL-10, and TNF-α were detected in tears and conjunctivas of operated eyes. Hematoxylin and eosin staining showed cornea neovascularization in the corneal stroma with thickened stroma layer and disorganized collagen bundles. Periodic acid-Schiff staining revealed a reduced function of conjunctival goblet cells. A combined type of DED model on the Beagle dog was established by removal of the orbital lacrimal gland and resection of the third eyelid. This DED model is easily accessible and is stable at six-month observation. The surgery-induced Beagle dog DED model is easily accessible and stable over a relatively long time.